Xylanase process for producing the same method for the treatment of pulp and production of xylo-oligosaccharides

ABSTRACT

Disclosed are novel xylanases, a process for producing the enzyme, a microorganism capable of producing the enzyme, a method for the treatment of pulp with the xylanase enzyme, and a process for producing xylose or xylo-oligosaccharide using the enzyme.

TECHNICAL FIELD

The present invention relates to a novel xylanase, a process forproducing the same, a microorganism producing the enzyme, a method forthe treatment of pulp with the xylanase, and a process for producingxylose or xylo-oligosaccharide using the enzyme.

BACKGROUND ART

A xylanase is an enzyme which hydrolyzes xylan or xylan polysaccharidesmainly composed of β-1,4-bonded-xyloses to yield its constituents,xylose and xylo-oligosaccharide. The xylanase is present widely inanimals and plants. Some microorganisms can also produce xylanases. Sofar investigations regarding xylanase-producing microorganisms have beenmade on bacteria, actinomycetes, yeasts, molds and the like.

In these years, keen attention has been brought to the use of xylanasesin biomass treatment. More specifically, xylanases are used in enzymaticbreakdown of agricultural wastes for production of alcoholic fuels, anenzymatic treatment of animal feeds to release free sugars, an enzymatictreatment for dissolving pulp in the preparation of cellulose and anenzymatic treatment in biobleaching of pulp. In particular, xylanase hasbeen highly expected in the paper and pulp industry wherein xylanesesare used to enhance the brightness of bleached pulp, improve the qualityof pulp, decrease the amount of chlorine used in the chemical pulpbleaching steps, and to increase the freeness of pulp in the recycledpaper process.

Turning to xylose, this compound is a product of xylan hydrolysis by axylanase and widely used as a raw material of foods and drugs.Xylo-oligosaccharide which is also a product of xylan hydrolysis isexpected to be for use as a sweetener or a moisturizer.

For the use in the foregoing purposes, it is desired to prepare axylanase suitable for mass production from inexpensive raw materials andhaving stability to an acid, an alkali and/or heat. Up to date, however,such a xylanase has not been achieved to meet the above requirements.

DISCLOSURE OF INVENTION

An object of the present invention is to provide a novel xylanase whichis thermally stable and acts stably over a wide pH range, a process forproducing the xylanase, a microorganism producing the xylanase, a methodfor the treatment of pulp with the xylanase, and a process for producingxylose or xylo-oligosaccharide using the xylanase.

The present inventor has made extensive investigations on enzymes andmicroorganisms suitable for the production of a desired xylanasesatisfying the requirements as stated above. As a result, it has beenfound that a novel microorganism belonging to the genus Bacillus canproduce a novel xylanase having physicochemical properties hithertounknown. The present invention has thus been attained.

Accordingly, a first aspect of the present invention is xylanase 1having the following physicochemical properties:

(1) acts on xylan or a xylan polysaccharide to hydrolyze theβ-1,4-xylosidic linkages in the molecule to yield xylose and xylobiosein a large quantity but to yield only in a small quantity axylo-oligosaccharide having a polymerization degree of at least that ofxylotriose;

(2) is active in the pH range of higher than 4.0 and lower than 10.0 andhas the optimum pH of about 6.0;

(3) is active in the temperature range up to 90° C. and has the optimumtemperature of about 75° C.;

(4) has a molecular weight of about 34,000 when determined by SDSpolyacrylamide gel electrophoresis; and,

(5) has an isoelectric point of about 9.4.

A second aspect of the present invention is xylanase 2 having thefollowing physicochemical properties:

(1) acts on xylan or a xylan polysaccharide to hydrolyze theβ-1,4-xylosidic linkages in the molecule to yield xylose and to yield axylo-oligosaccharide in a large quantity;

(2) is active in the pH range of higher than 2.6 and lower than 9.6 andhas the optimum pH of about 6.0;

(3) is active in the temperature range up to 90° C. and has the optimumtemperature of about 65 to about 70° C.;

(4) has a molecular weight of about 21,000 when determined by SDSpolyacrylamide gel electrophoresis; and,

(5) has an isoelectric point of about 9.8.

A third aspect of the present invention is a process for producing axylanase 1 or 2 which comprises culturing a microorganism belonging tothe genus Bacillus and recovering the xylanase 1 or 2 from the culturemedium.

A fourth aspect of the present invention is Bacillus sp. SD902 (FERMBP-4508), mutants thereof, or variants thereof obtained by Geneticengineering.

A fifth aspect of the present invention is a method for treating pulpwhich comprises acting a xylanase 1 or 2.

A sixth aspect of the present invention is a method for producing xyloseor a xylo-oligosaccharide which comprises acting a xylanase 1 or 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows graphs illustrating pH ranges in which xylanase 1 andxylanase 2 are active.

FIG. 2 shows graphs illustrating temperature ranges in which xylanase 1and xylanase 2 are active.

FIG. 3 shows graphs illustrating pH ranges in which xylanase 1 andxylanase 2 are stable.

FIG. 4 shows graphs illustrating temperature ranges in which xylanase 1and xylanase 2 are stable.

BEST MODE FOR CARRYING OUT THE INVENTION

The microorganism used in the present invention is bacteria belonging tothe genus Bacillus and capable of producing xylanase 1 and xylanase 2.The bacteria are isolated and obtained, for example, by the followingprocedures.

Firstly, soil samples collected from various places in Japan werecultured in a thermostat at 55° C. for 2 or 3 days on agar medium havingcompositions shown in Table 1 below.

                  TABLE 1    ______________________________________    Composition of Medium    ______________________________________    Oat xylan             0.5%    Asparagine            0.1%    Yeast extract         0.1%    Dipotassium hydrogenphosphate                          0.05%    Magnesium sulfate     0.02%    Sodium chloride       0.02%    Iron Sulfate          0.002%    ______________________________________

Bacteria colonies around which clear plaque (i.e., hollow) appeared wereisolated. Then, the strains obtained from the respective bacterialcolonies were cultured for 2 days, respectively, in liquid media havingcompositions shown in Table 2 below, each of which was charged in athermostat shaker at 55° C.

                  TABLE 2    ______________________________________    Composition of Medium    ______________________________________    Birch xylan           1.0%    Polypeptone           1.0%    Yeast extract         0.1%    Dipotassium hydrogenphosphate                          0.5%    Magnesium sulfate     0.05%    Sodium chloride       0.05%    Iron Sulfate          0.002%    ______________________________________

A xylanase activity of the culture broth was determined by the followingmethod to select a strain showing a potent activity.

The xylanase activity was determined as follows. An enzyme solution, 0.2ml, was mixed with 0.8 ml of 0.1M phosphate buffer (pH 7.0) containingas substrate 1.25% birch xylan (xylan derived from birch) followed byreacting them at 50° C. for 10 minutes. The reducing sugars formed weredetermined by the 3,5-dinitrosalicylate method. The xylanase activitydescribed hereinafter was also determined as described above. One unit(U) is defined as the amount of enzyme that produces 1 μmol of xylose inone minute.

One of the strains thus isolated and purified is Bacillus sp. SD902identified as described hereinafter.

The strain thus isolated, selected and purified possesses thebacteriological properties as described hereinafter, and has thus beenidentified as belonging to the genus Bacillus. This strain was calledBacillus sp. SD902 and was deposited with National Institute ofBioscience & Human-Technology of Industrial Science and Technology(Ibaraki-ken, Japan) on Dec. 25, 1992 and received FERM P-13356 as anaccession number. Then, the deposition was transferred into aninternational deposition under the Budapest Treaty on Dec 22, 1993, andreceived FERM BP-4508 as an accession number.

The thus obtained strain, Bacillus sp. SD902, was grown at 55° C. andits bacteriological properties were examined according to the proceduresdescribed in The Genus Bacillus (1973) and Bergey's Manual of SystematicBacteriology (1984).

(1) Morphological characteristics

(a) Shape and size of cell: a shape of rod, size of approximately0.4-0.8 μm×1.5-3.0 μm

(b) Polymorphism: none

(c) Mobility: mobile

(d) Spore formation: spore formed, a shape of ellipse, central tosubterminal location, a size of spore of approximately 0.5-0.8μm×0.8-1.3 μm

(e) Gram staining: positive

(2) Cultural growth condition in the following media

(a) Bouillon-agar plate culture:

It forms a yellowish white translucent circular colony with a flatsurface.

(b) Bouillon-agar slant culture:

It grows spreading

(c) Bouillon liquid culture:

Turbid

(d) Bouillon-gelatin stab culture: not liquefied

(e) Litmus milk:

Neither coagulated nor peptonized.

(3) Physiological properties

(a) Nitrate reduction: negative

(b) VP test: negative

(c) VP broth pH: 5.9

(d) Indole production: negative

(e) Hydrogen sulfide production: negative

(f) Starch hydrolysis: positive

(g) Pigment production: No pigment is produced.

(h) Catalase production: positive

(i) Oxidase production: positive

(k) Range of growth conditions:

It grows in a neutral pH range (pH of 6 to 8) and at a temperature offrom 25 to 60° C. but does not grow at 65° C.

(1) Behavior toward oxygen: obligate aerobic

(m) Denitrification reaction: negative

(n) utilization of inorganic nitrogen sources:

It utilizes ammonium salts but no nitrates.

(o) Urease: negative

(p) Utilization of citric acid: negative

(q) OF test:

It produces an acid anaerobically when glucose is used.

(r) Resistance to salt:

It grows in a NaCl concentration of 2.0%.

(t) Existence of production of acids from the following sugars:

    ______________________________________            1) L-arabinose                     ±            2) D-xylose                     +            3) D-glucose                     +            4) D-mannose                     +            5) D-fructose                     +            6) D-galactose                     -            7) maltose                     +            8) sucrose                     ±            9) lactose                     +           10) trehalose                     ±           11) D-sorbitol                     +           12) D-mannitol                     +           13) inositol                     +           14) glycerine                     +           15) starch                     -    ______________________________________

Based on the foregoing bacteriological properties, taxonomical searchwas made on the strain SD902 according to Bergey's Manual of SystematicBacteriology (1984). Thus, it is considered that this strain belongs tothe genus Bacillus in view of aerobic, gram-positive rods that formspore.

Further in view of the fact that the strain grows at temperatures above55° C., it is considered that the strain is akin to B.stearothermophilus, B. schelegelii, B. acidocaldarius, B. licheniformis,B. coagulans, B. brevis, etc. However, the properties of anaerobicgrowth, VP test, VP broth pH and growth temperature range shown in Table3 below reveal that strain SD902 has bacteriological propertiesdissimilar to any of these known standard bacteria belonging to thegenus Bacillus.

Therefore, it is concluded based on the current taxonomy that the strainSD902 should be considered to be a new strain belonging to the genusBacillus.

                                      TABLE 3    __________________________________________________________________________                                      stearo-               acido-        licheni- thermo-           SD902               caldarlus                    brevis                        coagulans                             formis                                 schlegelli                                      philus    __________________________________________________________________________    Anaerobic           -   -    -   +    +   -    -    growth    VP test           -   -    -   +    +   -    -    pH of VP           5.9 ND   8.0-8.6                        4.2-4.8                             5.0-6.5                                 ND   4.8-5.8    broth    Growth 25-60               50-65                    10-60                        15-60                             15-55                                 55-65                                      30-75    temperature    °C.    Growth at           -   ± ±                        +    +   -    -    pH 5.7    Gelatin           -   ND   ±                        -    +   -    +    liquefaction    Assimilation           +   +    -   +    +   -    ±    of starch    __________________________________________________________________________

The microorganism used in the present invention is not limited to theaforementioned strain SD902 and any strain may be used so far as it hasan ability of producing a xylanase having the properties describedhereinbelow. The strain Bacillus sp. SD902 may include its spontaneousand artificial mutants, and genetically engineered variants.

Artificial mutants of Bacillus sp. SD902 can be obtained by aconventional method. For example, an original strain is subjected toartificial mutation treatment such as irradiation with ultraviolet raysor treatment with a chemical, e.g., N-methyl-N'-nitro-N-nitrosoguanidine(NTG) and then planted on an agar medium containing Oat xylan andcultivated to grow colonies. The colonies are isolated, and are thencultivated on a conventional medium for xylanase production, and theresulting xylanase is checked for identify. Thus, a strain having themost excellent productivity for the objective xylanase can be screened.

Also, genetically engineered strains can be obtained by a conventionalmethod. For example, a presumption on DNA base sequence of thechromosome of the original strain is made based on amino acid sequenceof xylanase produced by the original strain, and a characteristicportion of the presumed DNA base sequence is synthesized. Then,phosphorus atoms in the phosphoric acid groups in the sequence arelabelled with radioisotope ³² p. On the other hand, the entirechromosomal DNA is extracted from the original strain and digested witha suitable restriction enzyme to obtain DNA fragments, which are thensubjected to Southern hybridization method to allow the chromosomalfragments to hybridize with the synthetic DNA. Thus, a chromosomalfragment which hybridizes with the synthetic DNA is screened.

The chromosomal fragment thus obtained is incorporated in a suitablevector and introduced in a xylanase non-producing strain and productionof xylanase is checked. The DNA fragment of which xylanase productionhas been confirmed is introduced in the original strain or a strainhaving a higher enzyme productivity (i.e., having a higher ability ofsecreting proteins) using a suitable vector such as a plasmid to obtaina strain of which productivity has been improved.

The strain, Bacillus sp. SD902, produces xylanase 1 and xylanase 2 whichare novel xylanases. For example, Bacillus sp. SD902 is inoculated onliquid medium containing xylan, yeast extract and polypeptone as themajor components and incubated at 55° C. for 1 to 3 days. The culturedmedium is centrifuged to remove the cells and insoluble matters. Theresulting culture supernatant is subjected to salting-out with ammoniumsulfate, ion exchange chromatography, gel filtration chromatography andthe like, in a conventional manner to isolate and/or purify xylanase 1and xylanase 2. Xylanase 1 and xylanase 2 can be isolated from eachother by, e.g., dialyzing the precipitates obtained after salting-outwith ammonium sulfate, then purifying the dialysate by anionic ionexchange chromatography and cationic ion exchange chromatography andfinally by gel filtration chromatography.

Physicochemical properties of xylanase 1 according to the presentinvention are listed below.

(1) Activity and substrate specificity:

The enzyme acts on xylan or xylan polysaccharide and hydrolyzes theβ-1,4-xylosidic linkages in the molecule to yield xylose and xylobiosein large quantities but yield only in a small quantityxylo-oligosaccharides having a polymerization degree of at least that ofxylotriose. Xylanase 1 does not have any substantial avicelase activityfor degrading crystalline cellulose or any substantial CMCase activityfor breakdown of carboxymethyl cellulose.

(2) Acting pH and the optimum pH:

When the xylanase activity is determined at 60° C. using acetate bufferor borate buffer having various pH values of from 2.5 to 10.0, xylanase1 acts in the pH range of higher than 4.0 and lower than 10.0 and hasthe optimum pH at about 6.0, as shown in FIG. 1. The enzyme shows morethan 70% activity of the maximum activity in the pH range of about 4.7to about 8.0, as shown in FIG. 1.

(3) Acting temperature and the optimum temperature:

When the xylanase activity is determined at various temperatures rangingfrom 30° C. to 90° C. using phosphate buffer (pH 7.0), xylanase 1 actsin the temperature range up to 90° C. and has the optimum temperature atabout 75° C., as shown in FIG. 2.

(4) pH stability:

When the xylanase activity is determined after maintaining at 40° C. for48 hours in acetate buffer, phosphate buffer or borate buffer havingvarious pH values in the range of 3.2 to 9.9, the enzyme retains morethan 95% of the activity in the pH range of 5.0 to 9.9, as shown in FIG.3.

(5) Thermal stability:

When the xylanase activity is determined after maintaining at 60° C. for24 hours in phosphate buffer (pH 7.0), the enzyme retains more than 90%of the activity, as shown in FIG. 4.

(6) Molecular weight:

The molecular weight is about 34,000 based on SDS polyacrylamide gelelectrophoresis.

(7) Isoelectric point:

The enzyme has the isoelectric point at pH of about 9.4.

Physicochemical properties of xylanase 2 according to the presentinvention are listed below.

(1) Activity and substrate specificity:

The enzyme acts on xylan or xylan polysaccharide and hydrolyzes theβ-1,4-xylosidic linkages in the molecule to yield xylose and also yieldin a large quantity xylo-oligosaccharides such as xylobiose, xylotriose,xylotetraose, etc. Xylanase 2 does not have any substantial avicelaseactivity for degrading crystalline cellulose or any substantial CMCaseactivity for breakdown of carboxymethyl cellulose.

(2) Acting pH and the optimum pH:

When its xylanase activity is determined at 60° C. using acetate bufferor borate buffer having various pH values of from 2.6 to 9.6, xylanase 2acts in the pH range of higher than 2.6 and lower than 9.6 and has theoptimum pH at about 6.0. The enzyme shows more than 70% activity of themaximum activity in the pH range of about 5.0 to about 7.7.

(3) Acting temperature and the optimum temperature:

When the xylanase activity is determined at various temperatures rangingfrom 30° C. to 90° C. using phosphate buffer (pH 7.0), xylanase 2 actsin the temperature range up to 90° C. and has the optimum temperature atabout 65° C. to 70° C.

(4) pH stability:

When the xylanase activity is determined after maintaining at 40° C. for48 hours in acetate buffer, phosphate buffer or borate buffer havingvarious pH values in the range of 3.2 to 9.9, the enzyme retains morethan 95% of the activity in the pH range of 4.0 to 9.0.

(5) Thermal stability:

When the xylanase activity is determined after maintaining at 60° C. for24 hours in phosphate buffer (pH 7.0), the enzyme retains more than 70%of the activity.

(6) Molecular weight:

The molecular weight is about 21,000 based on SDS polyacrylamide gelelectrophoresis.

(7) Isoelectric point:

The enzyme has the isoelectric point at pH of about 9.8.

Xylanase 1 or xylanase 2 can be produced by culturing Bacillus sp.SD902, mutants thereof, or variants thereof by genetic engineering, andrecovering the produced xylanase 1 or xylanase 2 from the culture broth.

For culturing, conventional methods of culturing bacteria may be usedand typical examples include liquid culture and solid culture. Amongothers, aerobic liquid culture is preferable from an economic viewpoint.Representative examples of such culturing are aerial spinner culturingand shake culturing, under aerobic conditions.

Any medium may be employed for the production of xylanases of thepresent invention so long as the strain used can proliferate. As carbonsources, carbonaceous compounds that can be assimilated or thosecontaining the same may be used, for example, various xylans; variousraw materials containing xylan or xylan polysaccharides such as wheatbran, pulp wastes, saccharified crop lees or rice straws; glucose,starch or starch hydrolysates such as liquefied starch; sugars such asmolasses, solely or as admixture thereof.

As nitrogen sources, there may be used nitrogen compounds that can beassimilated or those containing the same; for example, there may beused, singly or in combination, organic nitrogen-containing compoundssuch as various amino acids, corn steep liquor, maltose extract,peptone, soybean powders and defatted soybean powders; and inorganicnitrogen compounds such as ammonium salts, e.g., ammonium chloride andammonium sulfate.

Media may also appropriately contain other additives, if necessary, suchas various organic and inorganic materials required for growth of thebacteria and for production of the enzymes, or compounds containingthese materials, e.g., salts such as phosphates, magnesium salts,calcium salts and manganese salts; vitamins, yeast extract, and thelike.

The temperature for incubation may generally be in the range of 10 to70° C., and preferably in the range of 25 to 60° C., more preferably inthe range of 35 to 55° C., for the strain sp. SD902. The pH forincubation may generally be in the range of 4 to 10 throughout theentire phase of incubation but preferably in the range of 6 to 8 for thestrain sp. SD902. The time period for incubation is generally for 10 to120 hours. The incubation is stopped at the time when the amount ofxylanases accumulated reaches the maximum. For the strain sp. SD902, thetime period from 20 to 80 hours is preferable particularly from aneconomic aspect.

After completion of the incubation, xylanase 1 or xylanase 2 can berecovered from the culture broth in a conventional manner. That is, theculture broth is appropriately subjected to conventional operationsselected from precise filtration such as filter press filtration,membrane filtration, centrifugation, ammonium sulfate precipitation,concentration through membrane, drying, etc. to remove unnecessarymatters from the culture broth. Then xylanase 1 or xylanase 2 can beharvested as the enzyme solution or in the form of powders. For example,the cells in the culture broth are removed by centrifugation. Theresulting supernatant can be used as it is. Alternatively, thesupernatant may be diluted or concentrated appropriately, or added witha stabilizer and the resulting solution may be used as the enzymesolution. Further alternatively, the enzyme is precipitated from thesupernatant with, e.g., 60% ammonium sulfate and the precipitates arefiltered in a conventional manner to obtain the enzyme. The thusobtained enzyme may be dialyzed overnight and the dialysate is appliedto various ion exchange chromatographies or gel filtrationchromatography in a conventional manner to obtain xylanase 1 or xylanase2.

Xylanase 1 and xylanase 2 of the present invention do not substantiallypossess the cellulase activity for hydrolyzing cellulose as the majorconstituent of pulp. Accordingly, these enzymes are suitable to enhancethe brightness of pulp, to improve the quality of paper, to decrease theamount of chemical bleaching agents such as chlorine used in the pulpbleaching stages, and to treat pulp for other purposes, without inducingany damage of cellulose in pulp.

Where xylanase 1 or xylanase 2 is used for these pulp treatments, pulpis treated with the enzyme in the amount of 0.01 to 1000 U/g dry pulp,preferably 0.05 to 10 U/g dry pulp.

In the pulp treatment according to the present invention, conditions ofthe enzymes for treating pulp, such as temperature, pH, pressure, timeperiod, etc., may be suitably chosen so that the desired enzymaticaction is exhibited to achieve the desired effects such as enhancementof the brightness. For example, the temperature may be in the range of10 to 80° C., preferably 40 to 70 ° C. The pH may be in the range of 3to 10, preferably 4 to 9, more preferably 5 to 8.

The pressure may be applied under such a pressure conventionally usedfor pulp bleaching or other ordinary pulp treating steps; there is noparticular restriction but normal pressure is preferably from aneconomic standpoint. The time period for the treatments may be in therange of 10 minutes to 50 hours, preferably 1 to 24 hours, morepreferably 1 to 5 hours.

As will be shown below in the Examples, when unbleached Kraft pulp frombroadleaf trees is treated with 500 U/kg dry pulp of xylanase derivedfrom Bacillus sp. SD902, the lignin contained in the pulp is removed inlarge amounts by the treatment in a relatively short period of time.Furthermore, in the case where it is desired to enhance the brightness,the amount of a chemical bleaching agent used after the enzymatictreatment can be greatly reduced. It is considered that the pulptreatment of the present invention which provides the effects describedabove is sufficient as a substitute for at least a part of the currentbleaching process using chlorine bleaching agents.

The method of the present invention for treating pulp is applicable to awide range of pulp derived from a broadleaf tree, a needle-leaf tree ora non-tree material, such as kraft pulp, sulfite pulp, semi-chemicalpulp, groundwood pulp, refiner groundwood pulp, thermo-mechanical pulp,etc. By applying the pulp treatment method of the present invention tothese pulps, the amount of lignin remained in pulp can be reduced toattain the effects such as enhancement of the brightness of pulps,improvement of the quality, and decrease of the amount of a chemicalbleaching agent. The pulp treatment method of the present invention mayalso be applied to the bleaching steps of these pulps by oxygen or thelike, prior to or after the bleaching.

Following the pulp treatment using the xylanases of the presentinvention, an extraction may also be carried out to effectively removethe lignin dissolved or susceptible to be dissolved out of the pulp. Theextraction may be performed using, e.g., sodium hydroxide. In this case,typical conditions for the extraction are set forth to have a pulpconcentration of 0.3 to 20%, a sodium hydroxide concentration of 0.5 to5% based on the weight of dry pulp, a temperature range of 40 to 80° C.,and a time period for 30 minutes to 3 hours, preferably for 1 to 2hours.

After pulp is treated according to the method of the present invention,a chemical bleaching agent may also be used to further enhance thebrightness of the pulp. In this case, even if the amount of the chemicalbleaching agent is greatly decreased as compared to the case ofbleaching pulp only with the chemical bleaching agent, a betterbrightness can be obtained. Where chlorine dioxide is used as a chemicalbleaching agent, its amount can be reduced by 23% to 43% or even more.

When paper is made from the pulp so treated according to the method ofthe present invention, it has been found that the paper has excellentproperties such as a lower content of chlorinated phenol compounds, ascompared to paper prepared from conventional bleached pulp. It isconsidered to be because xylan, which is one of the hemicellulosecomponents in pulp, would be efficiently removed by the treatment withthe xylanase having no cellulase activity. However, the details of themechanism are yet unknown.

The present invention also provides a method for producing xylose orxylo-oligosaccharides such as xylobiose, xylotriose, xylotetraose andthe like which comprises acting xylanase 1 or xylanase 2 on xylan orxylan polysaccharides.

According to the method of the present invention, xylose orxylo-oligosaccharides may be produced using xylanase 1 or xylanase 2,under such conditions that the enzyme retains the xylanase activity. Forexample, the temperature for the treatment is in the range of about 10°to about 80° C., preferably 40° to 70° C.; the pH for the treatment isin the range of 3 to 10, preferably 4 to 9, more preferably 5 to 8. Thereaction time is set to such a period that the enzyme acts on xylan orxylan polysaccharides, for example, in the range of 10 minutes to 24hours, preferably 1 to 10 hours, more preferably 1 to 3 hours.

As the xylan or xylan polysaccharides used to produce xylose orxylo-oligosaccharides according to the present invention, there may beused agricultural wastes, e.g., rice straws, bagasse, wheat bran, corncob; woods, or xylans previously isolated therefrom. These xylan orxylan polysaccharides may be used in a concentration of 0.1 to 50%,preferably 0.5 to 30% in weight.

Xylanase 1 and xylanase 2 of the present invention do not substantiallypossess the cellulase activity which hydrolyzes cellulose. Accordingly,in the case of producing xylose or xylo-oligosaccharides, the enzyme isused in the range of 0.01 to 1000 U/g dry xylan, preferably 0.05 to 10U/g dry xylan.

The present invention is described in more detail by referring to theexamples. However, these examples are merely illustratively shown butthe present invention is not deemed to be limited the examples.

EXAMPLE 1

In a 5-liter volume jar fermenter was charged 2 liters of liquid medium(pH 7.0) composed of 1.0% birch xylan, 0.1% yeast extract, 1.0%polypeptone, 0.5% dipotassium hydrogenphosphate, 0.05% magnesiumsulfate, 0.002% iron sulfate and 0.05% sodium chloride, which was thensterilized at 121° C. for 20 minutes. The cells of Bacillus sp. SD902cultured in 100 ml of the same medium were inoculated on the liquidmedium followed by aerobic stirred culturing at 55° C. for 48 hours withstirring at a stirring speed of 1000 rpm in an aerial amount of 1liter/min. After the incubation, the culture broth was centrifuged at6000 rpm to remove the cells.

Ammonium sulfate was then added to 700 ml of the resulting supernatantto reach the 60% saturation for salting-out. The enzyme precipitatesobtained by the salting-out were dissolved in 50mM phosphate buffer (pH7.0). The solution was dialyzed overnight. After the dialysis, theenzyme solution was applied to anionic ion exchange chromatography(DEAE-Cellulofine, manufactured by Seikagaku Kogyo K. K.). The fractionpassed through was then applied to cationic ion exchange chromatography(CM- Cellulofine, manufactured by Seikagaku Kogyo K. K.). Thereafterelution was performed at a linear gradient of 1000 ml sodium chloride of0 to 0.6M to fractionate by 7 ml each. The fraction at which thexylanase activity was noted was collected and concentrated byultra-filtration. From the concentrate, 10 ml was taken and purified bygel filtration chromatography (Toyo Pearl HW55s, manufactured by TosoCo., Ltd.) using 5 mM phosphate buffer (pH 7.0) as an eluent. Xylanase 1(810 U) was obtained as the fraction eluted earlier and xylanase 2 (1150U) as the fraction eluted later.

EXAMPLE 2

To 0.5 wt % solution of birch xylan (manufactured by Sigma Inc., X-0502)was added xylanase 1 obtained in a manner similar to Example 1 in anamount of 200 U/g xylan. After pH was adjusted to 6.0, the mixture washeated to 60° C. to cause the reaction. The time course of the reactionwas monitored up to 24 hours. After the reaction was completed, thereaction mixture was treated at 100° C. for 5 minutes, the reactionmixture was centrifuged to remove the enzyme precipitates. The reactionproduct was analyzed by thin layer chromatography and high performanceliquid chromatography.

The analysis of the product by thin layer chromatography was performedas follows. The reaction product was developed on a thin layer(Kieselgel 60F254, manufactured by Merck Co.) with butanol: pyridine:water =8:1:1. As a color developer, a 5:1 mixture by volume of anacetone solution containing 0.2% diphenylamine and 0.2% aniline to 85%phosphoric acid was used. As the result of analysis, the production ofxylose and xylobiose was noted from the beginning of the reaction. Atthe later stage of the reaction, xylose and xylobiose produced weremarkedly increased.

The analysis of the reaction product by high performance liquidchromatography was performed as follows. The reaction product wasapplied to Shodex Ionpak S-801 (manufactured by Showa Denko K.K.) as agel filtration column for sugars, which was eluted with water forchromatographic treatment. The eluted component was detected with adifferential refractometer. The analysis of the product obtained afterthe reaction for 24 hours reveals that 22% (w/w) xylose and 40% (w/w)xylobiose were produced based on the starting birch xylan.

EXAMPLE 3

The reaction and analysis were conducted in a manner similar to Example2, except that 200 U/g xylan of xylanase 2 obtained in a manner similarto Example 1 was added to 0.5 wt % solution of birch xylan (manufacturedby Sigma, X-0502). As the result of analysis, the production of xyloseand xylobiose was noted from the beginning of the reaction. At the laterstage of the reaction, xylose and xylobiose produced were markedlyincreased. The analysis of the product obtained after the reaction for24 hours reveals that 8% (w/w) xylose, 34% (w/w) xylobiose and 7% (w/w)xylo-oligosaccharides having a polymerization degree of at least that ofxylotriose were produced based on the starting birch xylan.

EXAMPLE 4

The enzyme-containing supernatant prepared from the culture broth ofBacillus sp. SD902 obtained in a manner similar to Example 1 was addedto 500 g (50 g as dry pulp) of broadleaved unbleached kraft pulp slurryhaving a pulp concentration of 10 wt %, as the xylanase activity, in anamount of 100, 500 or 1000 U/kg dry pulp. In the enzymatic treatment,the pH was adjusted to 6. The pulp slurry was heated at 60° C. for anhour or for 3 hours. After filtering, the pulp was washed with 2-to3-fold amount of water. Thereafter water was added to the pulp so thatthe mixture was obtained to have a pulp concentration of 10 wt % and tocontain sodium hydroxide corresponding to 1.3 wt % based on the weightof dry pulp. The mixture was extracted at 60° C. for an hour followed bywashing the pulp with water.

An enzyme-free sample was prepared in a manner similar to the aboveexcept that no enzyme was added. The effect of the enzymatic treatmentwas compared with the sample. The kappa number used as an index oflignin content was determined according to the Japanese IndustrialStandards JIS P8211. The kappa numbers of the pulp obtained by thetreatments are shown in Table 4. The weight of dry pulp after theenzymatic treatment for 3 hours showed 49 g.

                  TABLE 4    ______________________________________    Time for    Enzymatic   Amount of Enzyme    Treatment   (U/kg pulp)   Kappa Number    ______________________________________    1            0            12.5    1           100           10.7    1           500           10.2    1           1000          10.0    3            0            12.5    3           100           10.5    3           500           10.1    3           1000          10.0    ______________________________________

EXAMPLE 5

The enzymatic treatment and extraction with sodium hydroxide werecarried out in a manner similar to Example 4 except for using 500 g (50g as dry pulp) of needle-leaved unbleached kraft pulp slurry having apulp concentration of 10 wt %. An enzyme-free sample was also preparedin a manner similar to Example 4. The kappa numbers of the pulp obtainedby these treatments are shown in Table 5. The weight of dry pulp afterthe enzymatic treatment for 24 hours showed 49 g.

                  TABLE 5    ______________________________________    Time for    Enzymatic   Amount of Enzyme    Treatment   (U/kg pulp)   Kappa Number    ______________________________________    1            0            21.8    1           100           19.8    1           500           19.6    1           1000          19.5    3            0            21.8    3           100           19.6    3           500           19.5    3           1000          19.4    ______________________________________

Comparative Example 1

Chlorine dioxide was added to broadleaved unbleached kraft pulp slurryhaving a pulp concentration of 10 wt % in 12.5 kg/t dry pulp. Themixture was heated at 70° C. for an hour. After the bleaching, themixture was washed with 2- to 3-fold amount of water. Then water wasadded to the pulp so that the mixture had a pulp concentration of 10 wt% and sodium hydroxide corresponding to 1.3 wt % based on the weight ofdry pulp was added. The mixture was extracted at 60° C. for an hour andthe pulp was then washed with water. The kappa number of the pulp was3.5.

Water was added to the thus obtained pulp extracted with sodiumhydroxide (kappa number of 3.5) so that the mixture was made to have apulp concentration of 10 wt % and to contain 18.9 kg of chlorinedioxide/t dry pulp. The mixture was heated at 70° C. for 3 hours and thepulp were then washed with 2- or 3-fold amount of water. The pulp wasdried and the bleached pulp had the Hunter brightness of 90%. The Hunterbrightness was determined by the Japanese Industrial Standards JISP8123. The total amount of chlorine dioxide used in the two-stagebleaching was 31.4 kg/t dry pulp.

EXAMPLE 6

The enzyme-containing supernatant prepared from the culture broth ofBacillus sp. SD902 obtained in a manner similar to Example 1 was addedto 500 g (50 g as dry pulp) of broadleaved unbleached kraft pulp slurryhaving a pulp concentration of 10 wt %, as the xylanase activity, in anamount of 500 U/kg dry pulp. In the enzymatic treatment, the pH wasadjusted to 6. The pulp slurry was heated at 60° C. for 3 hours. Afterfiltering, the pulp was washed with 2- to 3-fold amount of water.Thereafter water was added to the pulp so that the mixture was made tohave a pulp concentration of 10 wt % and to contain 10.2 kg of chlorinedioxide/t dry pulp. The mixture was heated at 70° C. for an hour. Thepulp was then washed with 2- or 3-fold amount of water. Water was thenadded to the pulp so that the mixture had a pulp concentration of 10 wt% and sodium hydroxide corresponding to 1.3 wt % based on the weight ofdry pulp was added. The mixture was extracted at 60° C. for an hour andthe pulp was then washed with water. The kappa number of the pulp was3.5.

Water was added to the thus obtained pulp (kappa number of 3.5)enzyme-treated, chlorine dioxide-treated and then sodiumhydroxide-extracted so that the mixture was made to have a pulpconcentration of 10 wt % and to contain 7.7 kg of chlorine dioxide/t drypulp. The mixture was heated at 70° C. for 3 hours and the pulp was thenwashed with 2- or 3-fold amount of water. The pulp was dried and thebleached pulp had the Hunter brightness of 90%. The total amount ofchlorine dioxide used in the two stage bleaching was 17.9 kg/t dry pulp.The amount of chlorine dioxide used was less by 43% than ComparativeExample 1 where no enzyme was used.

EXAMPLE 7

Xylanase 1 obtained in a manner similar to Example 1 was added to 500 g(50 g as dry pulp) of broadleaved unbleached kraft pulp slurry having apulp concentration of 10 wt %, in an amount of 500 U/kg dry pulp as thexylanase activity. In the enzymatic treatment, the pH was adjusted to 6.The pulp slurry was heated at 60° C. for 3 hours. After filtering, thepulp was washed with 2- to 3-fold amount of water. Thereafter water wasadded to the pulp so that the mixture was made to have a pulpconcentration of 10 wt % and to contain 10.1 kg of chlorine dioxide/tdry pulp. The mixture was heated at 70° C. for an hour. The pulp wasthen washed with 2- or 3-fold amount of water. Water was then added tothe pulp so that the mixture had a pulp concentration of 10 wt % andsodium hydroxide corresponding to 1.3 wt % based on the weight of drypulp was added. The mixture was extracted at 60° C. for an hour and thepulp was then washed with water. The kappa number of the pulp was 3.5.

Water was added to the thus obtained pulp (kappa number of 3.5)enzyme-treated, chlorine dioxide-treated and then sodiumhydroxide-extracted so that the mixture was made to have a pulpconcentration of 10 wt % and to contain 7.7 kg of chlorine dioxide/t drypulp. The mixture was heated at 70° C. for 3 hours and the pulp was thenwashed with 2- or 3-fold amount of water. The pulp was dried and thebleached pulp had the Hunter brightness of 90%. The total amount ofchlorine dioxide used in the two stage bleaching was 17.8 kg/t dry pulp.The amount of chlorine dioxide used was less by 43% than ComparativeExample 1 where no enzyme was used.

EXAMPLE 8

Xylanase 2 obtained in a manner similar to Example 1 was added to 500 g(50 g as dry pulp) of broadleaved unbleached kraft pulp slurry having apulp concentration of 10 wt %, in an amount of 500 U/kg dry pulp as thexylanase activity. The pulp slurry was heated at 60° C. for 3 hours.After filtering, the pulp was washed with 2- to 3-fold amount of water.Thereafter water was added to the pulp so that the mixture was made tohave a pulp concentration of 10 wt % and to contain 11.3 kg of chlorinedioxide/t dry pulp. The mixture was heated at 70° C. for an hour. Thepulp was then washed with 2- or 3-fold amount of water. Water was thenadded to the pulp so that the mixture had a pulp concentration of 10 wt% and sodium hydroxide corresponding to 1.3 wt % based on the weight Ofdry pulp was added. The mixture was extracted at 60° C. for an hour andthe pulp was then washed with water. The kappa number of the pulp was3.5.

Water was added to the thus obtained pulp (kappa number of 3.5)enzyme-treated, chlorine dioxide-treated and then sodiumhydroxide-extracted so that the mixture was made to have a pulpconcentration of 10 wt % and to contain 12.9 kg of chlorine dioxide/tdry pulp. The mixture was heated at 70° C. for 3 hours and the pulp wasthen Washed with 2- or 3-fold amount of water. The pulp was dried andthe bleached pulp had the Hunter brightness of 90%. The total amount ofchlorine dioxide used in the two state bleaching was 24.2 kg/t dry pulp.The amount of chlorine dioxide used was less by 23% than ComparativeExample 1 where no enzyme was used.

EXAMPLE 9

The enzyme-containing supernatant prepared from the culture broth ofBacillus sp. SD902 obtained in a manner similar to Example 1 was addedto 500 g (50 g as dry pulp) of broadleaved unbleached kraft pulp slurryhaving a pulp concentration of 10 wt %, as the xylanase activity, in anamount of 500 U/kg dry pulp. In the enzymatic treatment, the pH wasadjusted to 6. The pulp slurry was heated at 60° C. for 3 hours. Afterfiltering, the pulp was washed with 2- to 3-fold amount of water.Thereafter water was added to the pulp so that the mixture was made tohave a pulp concentration of 10 wt % and to contain 10.2 kg of chlorinedioxide/t dry pulp. The mixture was heated at 70° C. for an hour. Afterthe bleaching, the pulp was washed with 2- or 3-fold amount of water.Water was then added to the pulp so that the mixture was made to have apulp concentration of 10 wt % and to contain sodium hydroxidecorresponding to 1.3 wt % based on the weight of dry pulp. The mixturewas extracted at 60° C. for an hour followed by washing the pulp withwater. The kappa number of the pulp showed 3.5. On the other hand, pulpshowing the kappa number of 3.5 were obtained in a manner similar toComparative Example 1, using no enzyme.

Water was added to the thus obtained pulp having the kappa number of 3.5so that the mixture was made to have a pulp concentration of 10 wt % andto contain 8.0 kg of chlorine dioxide/t dry pulp. The mixture was heatedat 70° C. for 3 hours and the pulp was then washed with 2- or 3-foldamount of water. After drying, the Hunter brightness was determined. Asthe result, the Hunter brightness of the enzyme-treated pulp was 90.2%,whereas the Hunter brightness of the enzyme-untreated pulp was 88.5%.

EXAMPLE 10

Xylanase 1 obtained in a manner similar to Example 1 was added to 500 g(50 g as dry pulp) of broadleaved unbleached kraft pulp slurry having apulp concentration of 10 wt %, in an amount of 500 U/kg dry pulp as thexylanase activity. The pulp slurry was heated at 60° C. for 3 hours.After filtering, the pulp was washed with 2- to 3-fold amount of water.Thereafter water was added to the pulp so that the mixture was made tohave a pulp concentration of 10 wt % and to contain 10.1 kg of chlorinedioxide/t dry pulp. The mixture was heated at 70° C. for an hour. Thepulp was then washed with 2- or 3-fold amount of water. Water was thenadded to the pulp so that the mixture had a pulp concentration of 10 wt% and sodium hydroxide corresponding to 1.3 wt % based on the weight ofdry pulp was added. The mixture was extracted at 60° C. for an hour andthe pulp was then washed with water. The kappa number of the pulp was3.5. On the other hand, pulp showing the kappa number of 3.5 wasobtained in a manner similar to Comparative Example 1, using no enzyme.

Water was added to the thus obtained pulp having the kappa number of 3.5so that the mixture was made to have a pulp concentration of 10 wt % andto contain 8.0 kg of chlorine dioxide/t dry pulp. The mixture was heatedat 70° C. for 3 hours and the pulp was then washed with 2- or 3-foldamount of water. After drying, the Hunter brightness was determined. Asthe result, the Hunter brightness of the enzyme-treated pulp was 90.2%,whereas the Hunter brightness of the enzyme-untreated pulp was 88.0%.

EXAMPLE 11

Xylanase 2 obtained in a manner similar to Example 1 was added to 500 g(50 g as dry pulp) of broadleaved unbleached kraft pulp slurry having apulp concentration of 10 wt %, in an amount of 500 U/kg dry pulp as thexylanase activity. The pulp slurry was heated at 60° C. for 3 hours.After filtering, the pulp was washed with 2- to 3-fold amount of water.Thereafter water was added to the pulp so that the mixture was made tohave a pulp concentration of 10 wt % and to contain 11.3 kg of chlorinedioxide/t dry pulp. The mixture was heated at 70° C. for an hour. Thepulp was then washed with 2- or 3-fold amount of water. Water was thenadded to the pulp so that the mixture had a pulp concentration of 10 wt% and sodium hydroxide corresponding to 1.3 wt % based on the weight ofdry pulp was added. The mixture was extracted at 60° C. for an hour andthe pulp was then washed with water. The kappa number of the pulp was3.5. On the other hand, pulp showing the kappa number of 3.5 wasobtained in a manner similar to Comparative Example 1, using no enzyme.

Water was added to the thus obtained pulp having the kappa number of 3.5so that the mixture was made to have a pulp concentration of 10 wt % andto contain 12.0 kg of chlorine dioxide/t dry pulp. The mixture washeated at 70° C. for 3 hours and the pulp was then washed with 2- or3-fold amount of water. After drying, the Hunter brightness wasdetermined. As the result, the Hunter brightness of the enzyme-treatedpulp was 89.8%, whereas the Hunter brightness of the enzyme-untreatedpulp was 89.0%.

Industrial Applicability

According to the present invention, there are provided xylanase 1 andxylanase 2 which are novel enzymes, as well as the process for producingthe enzymes. These novel xylanases are effectively employed for biomasstreatments, e.g., to enhance the brightness of pulp, to improve thequality, to decrease the amount of a chemical bleaching agent in thepulp bleaching stages and to increase the freeness of pulp. The presentinvention also provides the method for such pulp treatments. By suchpulp treatments according to the present invention, large quantities ofthe lignin can be removed by the enzymatic treatment in a relativelyshort period of time, resulting in the enhanced brightness of pulp, theimproved quality, the decreased amount of a chemical bleaching agent,etc. The novel xylanases of the present invention are efficientlyutilized to prepare xylose or xylo-oligosaccharides widely used assweeteners, moisturizers, feeds, etc. in the food industry, cosmeticindustry and feed industry. According to the present invention, thereare also provided the method of preparing xylose orxylo-oligosaccharides. The present invention also provides the bacteriabelonging to the genus Bacillus that can produce these novel xylanases.

We claim:
 1. An isolated xylanase having the following physicochemicalproperties:(A) acts on a xylan molecule to hydrolyze β-1,4-xylosidiclinkages in the molecule to yield more xylose and xylobiose than axylo-oligosaccharide having a polymerization degree of at least that ofxylotriose; (B) is active in the pH range of higher than 4.0 and lowerthan 10.0 and has an optimum pH of about 6.0; (C) is active in atemperature range up to 90° C. and has an optimum temperature of about75° C.; (D) has a molecular weight of about 34,000 as determined by SDSpolyacrylamide gel electrophoresis; and (E) has an isoelectric point ofabout 9.4,wherein said xylanase is obtained by culturing Bacillus sp.SD902 (FERM BP-4508) or mutants having all the identifyingcharacteristics thereof, and recovering the xylanase from the culturemedium.
 2. An isolated xylanase having the following physicochemicalproperties:(A) acts on a xylan molecule to hydrolyze β-1,4-xylosidiclinkages in the molecule to yield xylose and a xylo-oligosaccharide; (B)is active in the pH range of higher than 2.6 and lower than 9.6 and hasan optimum. pH of about 6.0; (C) is active in a temperature range up to90° C. and has an optimum temperature of about 65 to about 70° C.; (D)has a molecular weight of about 21,000 as determined by SDSpolyacrylamide gel electrophoresis; and, (E) has an isoelectric point ofabout 9.8wherein said xylanase is obtained by culturing Bacillus sp.SD902 (FERM BP-4508) or mutants having all the identifyingcharacteristics thereof, and recovering the xylanase from the culturemedium.
 3. A process for producing an isolated xylanase which comprisesthe steps of:(A) culturing a microorganism belonging to the genusBacillus, and (B) recovering a xylanase having the followingphysicochemical properties: (a) (i) acts on a xylan molecule tohydrolyze β-1,4-xylosidic linkages in the molecule to yield more xyloseand xylobiose than xylo-oligosaccharide having a polymerization degreeof at least that of xylotriose; (ii) is active in the pH range of higherthan 4.0 and lower than 10.0 and has an optimum pH of about 6.0;(iii) isactive in a temperature range up to 90° C. and has an optimumtemperature of about 75° C.; (iv) has a molecular weight of about 34,000as determined by SDS polyacrylamide gel electrophoresis; and (v) has anisoelectric point of about 9.4, or recovering a xylanase having thefollowing physiochemical properties: (b)(i) acts on a xylan molecule tohydrolyze β-1,4-xylosidic linkages in the molecule to yield xylose and axylo-oligosaccharide; (ii) is active in the pH range of higher than 2.6and lower than 9.6 and has an optimum pH of about 6.0; (iii) is activein a temperature range up to 90° C. and has an optimum temperature ofabout 65 to about 70° C.; (iv) has a molecular weight of about 21,000 asdetermined by SDS polyacrylamide gel electrophoresis; and, (v) has anisoelectric point of about 9.8wherein said microorganism belonging tothe genus Bacillus is a Bacillus sp. SD902 (FERM BP-4508) bacteria andor mutants having all of the identifying characteristics thereof.
 4. Abiologically pure culture of Bacillus sp. SD902 (FERM BP-4508) ormutants having all of the identifying characteristics thereof.
 5. Amethod for treating pulp which comprises the step of contacting pulpwith an isolated xylanase having the following physicochemicalproperties:(a) (i) acts on a xylan molecule to hydrolyze β-1,4-xylosidiclinkages in the molecule to yield more xylose and xylobiose thanxylo-oligosaccharide having a polymerization degree of at least that ofxylotriose; (ii) is active in the pH range of higher than 4.0 and lowerthan 10.0 and has an optimum pH of about 6.0; (iii) is active in atemperature range up to 90° C. and has an optimum temperature of about75° C.; (iv) has a molecular weight of about 34,000 as determined by SDSpolyacrylamide gel electrophoresis; and (v) has an isoelectric point ofabout 9.4, orwith an isolated xylanase having the followingphysiochemical properties: (b)(i) acts on a xylan molecule to hydrolyzeβ-1,4-xylosidic linkages in the molecule to yield xylose and axylo-oligosaccharide; (ii) is active in the pH range of higher than 2.6and lower than 9.6 and has an optimum pH of about 6.0; (iii) is activein a temperature range up to 90° C. and has an optimum temperature ofabout 65 to about 70° C.; (iv) has a molecular weight of about 21,000 asdetermined by SDS polyacrylamide gel electrophoresis; and (v) has anisoelectric point of about 9.8,wherein said xylanase is obtained byculturing Bacillus sp. SD902 (FERM BP4508) or mutants having all theidentifying characteristics thereof, and recovering the xylanase fromthe culture medium.
 6. A method for producing xylose which comprises thestep of contacting xylan with an isolated xylanase having the followingphysicochemical properties:(a)(i) acts on a xylan molecule to hydrolyzeβ-1,4-xylosidic linkages in the molecule to yield more xylose andxylobiose than xylo-oligosaccharide having a polymerization degree of atleast that of xylotriose; (ii) is active in the pH range of higher than4.0 and lower than 10.0 and has an optimum pH of about 6.0; (iii) isactive in a temperature range up to 90° C. and has an optimumtemperature of about 75° C.; (iv) has a molecular weight of about 34,000as determined by SDS polyacrylamide gel electrophoresis; and (v) has anisoelectric point of about 9.4, orwith an isolated xylanase having thefollowing physiochemical properties: (b) (i) acts on a xylan molecule tohydrolyze β-1,4-xylosidic linkages in the molecule to yield xylose and axylo-oligosaccharide; (ii) is active in the pH range of higher than 2.6and lower than 9.6 and has an optimum pH of about 6.0; (iii) is activein a temperature range up to 90° C. and has an optimum temperature ofabout 65 to about 70° C.; (iv) has a molecular weight of about 21,000 asdetermined by SDS polyacrylamide gel electrophoresis; and (v) has anisoelectric point of about 9.8,wherein said xylanase is obtained byculturing Bacillus sp. SD902 (FERM BP-4508) or mutants having all theidentifying characteristics thereof, and recovering the xylanase fromthe culture medium.